Harmful Algae 5 (2006) 380–394 www.elsevier.com/locate/hal
Direct uptake of nitrogen by Pfiesteria piscicida and Pfiesteria shumwayae, and nitrogen nutritional preferences Patricia M. Glibert a,*, JoAnn M. Burkholder b, Matthew W. Parrow b, Alan J. Lewitus c,d,1, Daniel E. Gustafson a,2 a
University of Maryland Center for Environmental Science, Horn Point Laboratory, PO Box 775, Cambridge, MD 21613, United States b Center for Applied Aquatic Ecology, North Carolina State University, Raleigh, NC 27695, United States c Belle W. Baruch Institute of Marine Science and Coastal Research, University of South Carolina, Baruch Marine Laboratory, Georgetown, SC 29442, United States d Marine Resources Research Institute, South Carolina Department of Natural Resources, Hollings Marine Laboratory, Charleston, SC 29412, United States Received 5 December 2005; received in revised form 10 April 2006; accepted 28 April 2006
Abstract The rates of uptake of a range of forms of nitrogenous nutrients were measured in cultures of Pfiesteria piscicida and Pfiesteria shumwayae maintained at varying physiological states. The measured rates of dissolved N uptake under some conditions approached the rates of N uptake that are achieved through phagotrophy. Rates of dissolved N uptake by P. piscicida contributed urea > NH4+ > NO3. Nitrate consistently was not a preferred form of N. Although Pfiesteria spp. are often found in eutrophic environments, the relationship between Pfiesteria spp. and nutrient availability is likely to be primarily indirect, mediated through the production of various prey on which Pfiesteria spp. feed. These findings also confirm, however, that when dissolved N concentrations are elevated, they can contribute to the supplemental nutrition of these cells, and thus may provide a significant source of N to Pfiesteria spp. in nature. # 2006 Elsevier B.V. All rights reserved. Keywords: Pfiesteria; Nitrogen nutrition; Heterotrophy; Toxigenic dinoflagellates
1. Introduction * Corresponding author. Tel.: +1 410 221 8422; fax: +1 410 221 8290. E-mail address:
[email protected] (P.M. Glibert). 1 Present address: National Oceanic and Atmospheric Administration, 1305 East West Highway, Silver Spring, MD 20910, United States. 2 Present address: Chesapeake Research Consortium, 645 Contees Wharf Rd., Edgewater, MD 21037, United States. 1568-9883/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2006.04.009
Pfiesteria piscicida and Pfiesteria shumwayae (Marshall et al., in press) are heterotrophic dinoflagellates that feed phagotrophically on fish tissue, microalgae and other particles (Burkholder and Glasgow, 1997; Burkholder et al., 1998; Lewitus et al., 1999a,b). Nutrientenriched conditions have been correlated with Pfiesteria abundance (e.g. Burkholder and Glasgow, 1997; Lewitus
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et al., 1999b; Glibert et al., 2001, 2004), but the causes of this relationship are not fully understood. Unless Pfiesteria has the ability to use dissolved nutrients directly, the correlation with high nutrient loading would have to be mediated by the production of other organisms such as algal prey (Burkholder et al., 2001a,b). Although primarily phagotrophic, P. piscicida previously has been reported to take up some dissolved substrates, such as amino acids and protein hydrolysate (Burkholder and Glasgow, 1997). Moreover, Lewitus et al. (1999b) reported that P. piscicida has the ability to take up dissolved inorganic nitrogen (N) compounds, and that rates under nutrient-rich conditions may approach those of N acquisition through phagotrophy. Lewitus et al. (1999b) suggested that two pathways linking Pfiesteria with nutrient enrichment might occur: during spring, Pfiesteria may depend on phagotrophy to a greater extent when chlorophyll abundance is high, whereas during the summer when pathways of recycling dominant the flows of nitrogen, a direct pathway of N uptake may be more significant. Glasgow et al. (2001a) expanded this conceptual framework to include the role of fish prey. It is now well recognized that Pfiesteria spp., among many other autotrophic and heterotrophic dinoflagellate species, display considerable differences among species, strains, and even within the same strain when grown under different growth conditions (Burkholder et al., 2001a,b, 2005). For Pfiesteria spp. three operational terms have been used to describe different functional types (toxicity status) among the same strain or species. These have been identified as TOX-A, cells that are grown in the presence of live fish and can actively kill fish with toxin involvement; TOX-B, cells that had previously been grown under fish-killing conditions, then were removed from fish and grown on algal prey, still retaining the ability to kill fish with toxin when re-exposed; and non-inducible (NON-IND), cells that have been grown for extended periods on algal prey and have apparently lost the ability to kill fish with toxin upon re-exposure (Turgeon et al., 2001). NON-IND strains are the most common types collected from estuaries and maintained in culture, especially when grown for extended periods on algal prey (Burkholder et al., 2001a,b). Many differences in physiology and behavior of these functional types have previously been described, such as in response to fish, algal prey and inorganic nutrients (Burkholder et al., 2001a). Furthermore, Stoecker et al. (2002) and Lewitus et al. (in press) demonstrated that grazing by microzooplankton on TOX-A P. piscicida was significantly less than that of TOX-B and NON-IND functional types of the same strain.
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In this study we address the uptake of dissolved nitrogenous compounds by P. piscicida and P. shumwayae in several functional states. We also estimate the contribution of these forms of N to the potential growth of these species relative to heterotrophic consumption under nutrient-rich conditions. 2. Methods Two types of experiments were conducted. The first experiment (27–28 June 2000) was designed to investigate the range of N uptake rates by one clone of P. piscicida under differing growth conditions. The second experiment (12–13 December 2002) was designed to compare the rates of N uptake by P. piscicida and P. shumwayae grown under a range of nutritional and toxic conditions, and to assess the relationship between N uptake and substrate concentration in more detail. All cultures were clonal but not bacteria free. 2.1. Experiment 1 Pfiesteria piscicida (clone CAAE 416T) was originally collected from Middle River, Maryland (27 August 1999), and isolated using flow cytometric procedures (Burkholder et al., 2001a). Clonal status was cross-confirmed by the heteroduplex mobility assay (Oldach et al., 2000). Cultures were maintained at 23 8C on a 12-h:12-h L:D cycle at 80 mmol photons m2 s1. The general protocol for culturing Pfiesteria with fish in standardized bioassays was described by Burkholder et al. (2001b). Toxic cultures were fed 2–3 juvenile tilapia (Oreochromis mossambicus, length 5–7 cm) daily up to the time they were removed for the experiment. Three sub-cultures were used in this experiment. A TOX-A sub-culture was removed from fish cultures on the morning before the experiment. A TOX-B subculture was removed from fish cultures 2 weeks before the start of the experiment, recloned, and maintained on cryptophyte prey (Rhodomonas sp. CCMP757, Bigelow Laboratory Culture Collection). A NON-IND subculture was removed from fish cultures 10 months before the experiment and maintained on the same cryptophyte prey. Toxicity for all cultures was tested using the standardized bioassays of Burkholder et al. (2001b) and confirmed in the TOX-A and -B cultures by toxin analysis (Moeller et al., 2001; Burkholder et al., 2005). Twelve 100 ml aliquots from each culture type were dispensed into clean tissue culture flasks. Half of these
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sub-cultures were given Rhodomonas sp. as prey, while the other sub-cultures were not fed during the experimental period. The abundances of Rhodomonas, their change with time, and rate of grazing by Pfiesteria on this prey is the subject of a separate study (Lewitus et al., in press). Cell density for each sub-culture was estimated by light microscopy (400–600) using Lugol’s-preserved subsamples and a Palmer–Maloney counting chamber (Wetzel and Likens, 1991). The 12 flasks were then further divided so that equal numbers of flasks with and without prey were incubated under 560 mmol photons m2 s1 (hereafter referred to as ‘‘high light’’), and 12 mmol photons m2 s1 (hereafter referred to as ‘‘low light’’). All data reported here are the treatments without prey only. The treatments with prey were used in grazing studies (Lewitus et al., in press). Rates of uptake of various N substrates were then assessed for the treatments without prey on Day 1, and again 24 h later (Day 2). The rate of uptake of N by the control cultures of Rhodomonas sp. was assessed on Day 2. Rates of N uptake were determined by transferring 50 ml from each flask to a clean tissue culture flask and enriching with 15N substrates, either NH4+, NO3 or urea, at a final concentration of 40 mmol N L1. All 15N substrates were 97–99% enriched. The concentration of 15N added, 40 mmol N L1, represented from 5–70 at.% enrichment of ambient. As described below, data were rejected from further analysis when the initial enrichment was
